Stephen T. Haley writes:
>      I am trying to do some "relative" quantitative immunoflourescence
> on FITC labelled tissue sections using a Bio-Rad MRC-1000.
> I was happily collecting data.
> The levels of the surface markers I am trying to measure went up or
> down as I expected. The trouble arose when I tried to check the
> validity of my measurements with some InSpeck beads from Molecular
> Probes.  The beads are calibrated to several relative intensities. To
> make a long story short I found out that the system is not linear.

Any chance that spherical aberration is contributing to the non-linearity?  I.e.
are the test beads at different depths under the coverslip?

Another thought: any chance that the beads are photobleaching?

Martin Wessendorf PhD, Asst Professor                [log in to unmask]
Dept Cell Biology and Neuroanatomy                      (612) 624 2991 (voice)
Confocal Microscopy Facility                              (612) 624 8118 (FAX)
University of Minnesota
Minneapolis, MN  55455